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Method for treatment of diseases

a disease and disease technology, applied in the field of diseases, can solve the problems of vascular leakage into underlying tissues, edema causing fluid leakage into tissues, and can have serious and life-threatening consequences, and achieve the effect of reducing the od obtained and no effect on cell proliferation

Inactive Publication Date: 2012-03-29
AMPIO PHARMA INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0101]Additional objects, advantages and novel features of the present invention will become apparent to those skilled in the art by consideration of the following non-limiting examples.

Problems solved by technology

Dysregulation of this process produces vascular leakage into underlying tissues.
Leakage of fluid into tissues causing edema can have serious and life threatening consequences in a variety of diseases.

Method used

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  • Method for treatment of diseases
  • Method for treatment of diseases
  • Method for treatment of diseases

Examples

Experimental program
Comparison scheme
Effect test

example 1

Danazol's Effects on Angiogenesis (Comparative)

[0102]A. HUVEC Cell Proliferation

Protocol:

[0103]Primary human umbilical vein endothelial cells (HUVECs) and EGM-2 growth medium were obtained from Cambrex (Walkersville, Md.). The cells were passaged in medium supplemented with 2% fetal calf serum (FCS) in tissue culture flasks at 37° C. and 5% CO2. Subculturing was performed using trypsin when 60-80% confluence was obtained as specified by the supplier.

[0104]Cryopreserved ampoules of passage 2 HUVECs were thawed and plated in 96 well tissue culture plates at 5,000 cells / cm2. A 50 mM stock solution of danazol was prepared in ethanol and the FCS in the medium was increased to 5% to keep danazol in solution. The cells were treated with medium containing final concentrations of danazol ranging from 0.1 to 100 μM in triplicates. 24, 48, and 72 hour incubations were performed and cell proliferation was determined utilizing Celltiter 96 AQueous One Solution Cell Proliferation assay from Prome...

example 2

Danazol Effect on Vascular Permeability of HUVEC Monolayers

[0122]Protocol:

[0123]Assays were performed to determine the effect of danazol on permeability of HUVEC monolayers. Passage 5-10 HUVECs, lot number 7016 (obtained from Lonza), were seeded onto 1-micron-pore-size inserts located in the wells of a 24-well plate (Greiner BioOne 24-well Thincert cell culture inserts, #662610, or ISC BioExpress, #T-3300-15) using endothelial growth medium-2 (EGM-2) (obtained from Lonza). The plates were cultured in a 37° C. incubator with 5% CO2 for 48-72 hours to achieve confluence and develop tight monolayers. The medium was then removed and replaced with fresh medium or fresh medium containing a range of danazol concentrations (Sigma, #D8399). Tumor necrosis factor α (TNFα; Pierce Biotechnology, #RTNFAI) and interleukin-1β (IL-1β; Sigma, #I-9401) were added to appropriate wells at final concentrations of 10 ng / ml each. TNFα and IL-1β induce permeability; they can cause up to a ten-fold increase...

example 3

Danazol Effect on Vascular Permeability

[0127]Passage 9 human retinal endothelial cells (ACBR1181, Applied Cell Biology Research Institute, Kirkland, Wash.) were passaged in EGM-2 medium (Lonza, Walkersville, Md.) until 80% confluence was obtained. The cells were then released from the passage flask using Trypsin-EDTA, and the cells in the resulting suspension were counted to determine both viability and cell numbers. Viability of the cell suspension was greater than 90% in this experiment.

[0128]The cells were then seeded onto inserts (1 micron pore size) located in the wells of a 24-well plate (Greiner BioOne 24-well Thincert cell culture inserts, #662610) in 300 μl EGM-2 complete medium (obtained from Lonza). Then, 700 μl EGM-2 was placed in the bottom chamber, and the plates were cultured in a 37° C. incubator with 5% CO2 for 48 hours to achieve confluent monolayers. Transendothelial electrical resistance (TER) measurements were taken using an STX 100 electrode attached to EVOM2 v...

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Abstract

The invention provides a method of inhibiting vascular hyperpermeability in an animal in need thereof. The method comprises administering a vascular-hyperpermeability-inhibiting amount of a danazol compound to the animal. The invention also provides a method of modulating the cytoskeleton of an endothelial cell in an animal. The method comprises administering an effective amount of a danazol compound to the animal.

Description

[0001]This application claims benefit of provisional application 61 / 219,185, filed Jun. 22, 2009, and provisional application 61 / 315,350, filed Mar. 18, 2010, the complete disclosures of both of which are incorporated herein by reference.FIELD OF THE INVENTION[0002]The invention relates to a method of inhibiting vascular hyperpermeability and the edema and other adverse effects that result from it. The invention also relates to a method of modulating the cytoskeleton of endothelial cells. Both methods comprise administering a danazol compound to an animal.BACKGROUND[0003]The vascular endothelium lines the inside of all blood vessels. It acts as the interface between the blood and the tissues and organs. The endothelium forms a semi-permeable barrier that maintains the integrity of the blood fluid compartment, but permits passage of water, ions, small molecules, macromolecules and cells in a regulated manner. Dysregulation of this process produces vascular leakage into underlying tis...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K31/58A61P9/00C07J71/00
CPCA61K31/58A61P11/00A61P13/12A61P15/00A61P17/00A61P17/02A61P25/00A61P25/28A61P27/02A61P29/00A61P3/00A61P43/00A61P7/00A61P7/06A61P7/10A61P9/00A61P9/10A61P9/12A61P9/14A61P3/10A61K9/0053A61K2121/00
Inventor BAR-OR, DAVID
Owner AMPIO PHARMA INC
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